The macronucleus of hypotrichous ciliates contains "gene-sized" DNA molecules (average = 2,200 base pairs BP). There are about 24,000 different molecules, each present in an average of about 1,000 copies per macronucleus. The macronucleus divides amitotically, and there is no known mechanism by which the multiple copies of each gene can be equally distributed to daughter macronuclei. In addition, macronuclear division is variably unequal, distributing 0 to 12% more DNA to one macronucleus than the other (average = 4%). The presumed random distribution of genes and the unequal distribution of genes and the unequal distribution of DNA may lead to genetically imbalanced macronuclei, which would account for clonal senescence in ciliates. We propose to test this idea and two other possible mechanisms of aging in hypotrichs. 1) We will measure the distribution of DNA values for individual macronuclei in a clone. An increasing spread of values would be predicted from unequal macronuclear division. Lack of an increasing spread would imply a rectification mechanism for adjusting total DNA content. 2) We will test for developing imbalances in copy numbers of gene-sized molecules by densitometric scanning of DNA separated by size on agarose gels. We will also test for changes in copy numbers of genes coding for known products (rRNAs, tRNAs, snRNAs, histones, tubulins, and actins) as well as genes of unknown coding capacity by quantitative hybridization. 3) We plan to examine macronuclear DNA for accumulation of nicks and/or cross-links as a function of clonal aging. 4) We will test for reduction of total RNA complexity and reduction in amounts of specific RNAs during clonal aging.